Metabolic labeling of the bacterial peptidoglycan by functionalized glucosamine

Summary N-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) on N-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed by in vitro incorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolism in vivo.


INTRODUCTION
N-Acetylglucosamine (GlcNAc or NAG) is an essential monosaccharide required in almost all organisms. In bacteria, GlcNAc is a key component of several important cell envelope molecules, including lipopolysaccharide (LPS) (Raetz and Whitfield, 2002), peptidoglycan (PG) (Vollmer et al., 2008), wall teichoic acid (WTA) (Swoboda et al., 2010), enterobacterial common antigen (ECA) (Kuhn et al., 1988), and poly-N-acetylglucosamine (PNGA) (Roux et al., 2015). The PG layer is an essential component of bacteria that maintains the shape of the cell and protects it from bursting due to the high osmotic pressure inside the cytosol (Vollmer et al., 2008;de Pedro and Cava, 2015). The PG layer is composed of a polymer of alternating GlcNAc and N-acetylmuramic acid (MurNAc) sugars connected by short peptides attached to the lactoyl acid residue of MurNAc (de Pedro and Cava, 2015;Vollmer et al., 2008). The cytosolic pathway of PG biosynthesis leads to UDP-MurNAc-pentapeptide and UDP-GlcNAc, which on the inner leaflet of the plasma membrane are used for the formation of the final precursor of PG synthesis, undecaprenyl-pyrophosphoryl-MurNAc (pentapeptide)-GlcNAc (Figure 1), called Lipid II. Lipid II is then translocated across the cell membrane where it is used for PG synthesis (Mohammadi et al., 2011;Sham et al., 2014). The final stages of PG synthesis require two reactions: the polymerization of PG glycan chains by glycosyltransferase (GTase) reactions and the formation of the crosslinks between peptides on different chains by transpeptidase (TPase) reactions. Bifunctional class A penicillin-binding proteins (PBPs) can catalyze both GTase and TPase reactions ( Figure 1) (Sauvage et al., 2008;Sauvage and Terrak, 2016;Cho et al., 2016;Egan et al., 2017Egan et al., , 2020. The machinery involved in the biogenesis of the PG layer is an ideal target for antibiotics because of its essentiality and the absence of the pathway in mammalian cells. In recent years, several in vivo PG-labeling agents have been developed to investigate PG biosynthesis, elucidate the mode of action of antibiotics, and study interactions between pathogenic bacteria and their hosts (Hira et al., 2020). Further work developed procedures for preparing fluorescent D-amino acids, clickable D-amino acids, and D-amino acid dipeptides with bioorthogonal handles providing a means to study PG assembly and turnover in intact bacteria (Kuru et al., 2012(Kuru et al., , 2015Bisson-Filho et al., 2017;Monteiro et al., 2015Monteiro et al., , 2018Hsu et al., 2017;Hugonnet et al., 2016  iScience Article an azido group to form UDP-GlcNAz. The azido group would allow the specific labeling with probes via click chemistry. Since PBPs can accept MurNAc analogs of Lipid II as substrates for the synthesis of PG (DeMeester et al., 2018;Liang et al., 2017), we aimed to test if PBPs accept the GlcNAz version of Lipid II.
Traditional chemical synthesis of (unnatural) sugar-nucleotides has been reported over past decades (Ahmadipour and Miller, 2017;Timmons and Jakeman, 2007), whereas enzymatic synthesis has simplified access to these compounds. As previously demonstrated, different methods, some of which use multiple enzyme mixes, have provided access to the synthesis of sugar-nucleotides (Beaton et al., 2009;Guan et al., 2009Guan et al., , 2010Zhao et al., 2010;Chen et al., 2011;Masuko et al., 2012;Muthana et al., 2015;Li et al., 2016). Moreover, some glycosyltransferases (GTase)-catalyzed reactions are reversible (Zhang et al., 2006), making the formation of nucleotide diphosphate (NDP) sugars thermodynamically unfavorable (Zhang et al., 2006;Quiró s et al., 2000;Okada et al., 2009). Hence, it remains challenging to synthesize NDP-sugars from glycoside donors and nucleotide diphosphates. In 2011, Gantt and coworkers reported that aromatic O-glycosides can drastically shift the equilibrium of the GTase OleD-catalyzed reactions to overcome this thermodynamic barrier (Gantt et al., 2011). A variant of OleD (OleD TDP-16) was shown to catalyze the formation of NDP-sugars from a 2-chloro-4-nitrophenyl glycoside donor (Gantt et al., 2013). Moreover, this OleD variant was capable of catalyzing the formation of natural and non-natural UDP-activated sugars (Gantt et al., 2013;Zhang et al., 2014). Herein, we explore this approach for the synthesis of UDP-GlcNAz in one step by the enzyme OleD TDP-16. We demonstrate the synthesis of UDP-GlcNAz on a preparative scale and show that it is accepted as a substrate by Lipid II-synthesizing enzymes, enabling the efficient synthesis of GlcNAz-containing Lipid II, which was subsequently fluorescently labeled. We further show that this Lipid II variant was not accepted as a GTase substrate by PBPs which nevertheless incorporated fluorescently labeled GlcNAz-Lipid II into PG by TPase reactions. Finally, we succeeded in labeling the PG in Escherichia coli cells expressing OleD by feeding them with 2-chloro-4-nitrophenyl-N-azidoacetylglucosamine (GlcNAz-CNP) and subsequent analysis indicated that the labels were incorporated into the peptidoglycan layer.

RESULTS AND DISCUSSION
Chemical synthesis of azide-labeled D-glucosamine We first set out to optimize the synthesis of the activated sugar (GlcNAz-CNP) such that it would allow the facile synthesis of relatively large amounts. To synthesize an aromatic glycoside, the targeted hydroxyl group of the sugar needs to be activated by introducing a halide (Demchenko, 2008). In this way, the halide can be substituted with acceptors such as phenols to generate the desired glycoside. In a previous study, 2-chloro-4-nitrophenyl-b-D-glucopyranoside was synthesized using the reaction between a glycosyl bromide and 2-chloro-4-nitrophenol, where the glycosyl bromide was synthesized using a two-step procedure (Gantt et al., 2011). Here, we first introduce a chloroacetyl group on the amine of glucosamine to enable the installation of the azido group in the third step ( Figure 2A). In short, glucosamine hydrochloride was first neutralized by sodium methoxide (NaOMe) to give glucosamine. Then, the free amine of glucosamine was allowed to react with 2-chloroacetic anhydride to install a chloroacetyl group, that subsequently reacted with acetyl chloride, resulting in the protection of the hydroxyl groups at C3, C4, C6, and substitution at C1 to give compound 1. By using acetyl chloride as solvent and reaction substrate to produce compound 1, the acetylation and chloride-substitution are achieved in one step. The resulting product reacted with 2-chloro-4-nitrophenol in the presence of triethylamine to the 2-chloro-4-nitrophenyl glycoside. The final product, 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP), was synthesized by introduction of the azido group and removal of the acetyl groups.

Chemoenzymatic synthesis of UDP-GlcNAz
Next, we used the purified OleD variant TDP-16 for the synthesis of UDP-GlcNAz ( Figure 2B). For this, the enzyme was incubated with a mixture containing UDP, 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP), and MgSO 4 . The glycosyltransferase reaction was followed by measuring the appearance of yellow color due to the production of 2-chloro-4-nitrophenolate (CNP) during the reaction ( Figure S1E). The slight color in the control likely results from minor degradation of GlcNAc-CNP during the reaction. The reaction was also monitored by TLC which showed a new fluorescent spot under UV light (254 nm), indicative of the generation of a new product. This product was confirmed to be UDP-GlcNAz by MS ( Figure S1) and 31 P-NMR spectroscopy ( Figure 3A). The 13 C-and 1 H-NMR data of the purified UDP-GlcNAz product were consistent with previously reported data (Chen et al., 2011;Vocadlo et al., 2003;Debets et al., 2020)  iScience Article purification of the intermediates, and the isolation/purification of various enzymes, making it superior compared to standard chemical synthesis or multiple-enzyme-catalyzed processes (Chen et al., 2011;Vocadlo et al., 2003;Debets et al., 2020). Our method enables the easy installation of a bioorthogonal handle in UDP-GlcNAc, which is involved in multiple biosynthesis pathways and post-translational modifications in a multitude of organisms (Debets et al., 2020), making it easier to study these processes.

Synthesis of azido Lipid II and subsequent fluorescent labeling
The two most common variants of Lipid II differ at the third position in the pentapeptide. This residue is L-lysine in the Lipid II of most Gram-positive bacteria, and m-DAP in almost all Gram-negative bacteria. So far, in most fluorescently labeled Lipid II variants used in PG-synthesis assays, the fluorescent group was attached to the lysine at the third position of the pentapeptide (Mohammadi et al., 2011(Mohammadi et al., , 2014Breukink et al., 2003;Egan and Vollmer, 2016). This lysine is either directly used as an acceptor in PBP-catalyzed transpeptidation reactions or carries a branch made of L-amino acids that are used in cross-link formation. Hence, the presence of a fluorescent group at the lysine prevents these Lipid II variants from participating in transpeptidase reactions. To be able to measure the cross-linking in PG-synthesis assays, fluorescently labeled lysine Lipid II needs to be mixed with unlabeled Lipid II. We reasoned that Lipid II with a fluorescent label at the GlcNAc could circumvent this need for unlabeled Lipid II. Although MurG can convert Lipid I to Lipid II by utilizing UDP-GlcNAc in the positive control experiments, we failed to synthesize GlcNAzlabeled Lipid II from Lipid I by using purified MurG from E. coli under similar conditions (data not shown), indicating that this reaction may require different substrate concentrations or other conditions for optimal conversion to Lipid II. However, we could successfully synthesize GlcNAz-labeled Lys-or m-DAP-Lipid II by the reaction of UDP-GlcNAz and the corresponding type of Lipid I in a reaction catalyzed by a Micrococcus flavus membrane preparation (data not shown for Lys-Lipid II) ( Figures S2A and S2B). Next, we labeled the purified GlcNAz-labeled Lipid II (DAP) after CuAAC conjugation with dansyl alkyne. After further (B) One-step synthesis of UDP-GlcNAz by the glycosyltransferase (OleD) and its use in the synthesis of labeled Lipid II. The glycosyltransferase-catalyzed reaction between GlcNAz-CNP and UDP by OleD generated UDP-GlcNAz (step 1). UDP-GlcNAz was further utilized by MurG to attach GlcNAz to Lipid I (red oval), producing azide-labeled Lipid II (step 2).

PG synthesis with dansyl-GlcNAz-Lipid II
We next tested whether dansyl-GlcNAz-Lipid II was accepted by three class A PBPs from Gram-negative bacteria as a substrate in PG synthesis reactions ( Figure S3A). Reactions were carried out in the presence or absence of the respective outer membrane lipoprotein activators. Consumption of Lipid II and formation of glycan strands were monitored by SDS-PAGE (Herná ndez-Rocamora et al., 2021) (Figures 4 and S3). First, we tested E. coli PBP1B (PBP1B Ec ), which interacts with proteins from the cell division machinery and is a major contributor to the synthesis of septal PG ( One possible explanation for the inability of PBP1B to polymerize dansyl-GlcNAz-Lipid II was that this substrate is too bulky to simultaneously occupy the donor and acceptor sites at the GTase domain, as previously reported for Atto550-and Atto647-modified Lipid II variants ( Van't Veer et al., 2016). Therefore, we added an equal amount of unlabeled Lipid II to reactions to test if it allows reactions to proceed. We observed polymerized glycan strands at the front of the lane when using dansyl-GlcNAz-Lipid II ( . This indicates that the dansyl-GlcNAz-Lipid II was incorporated into PG by the transpeptidase activity of PBP1B, likely used as a donor with the growing chain being used as an acceptor. The amount of incorporation of dansyl-GlcNAz-Lipid II into PG was relatively low, which is in line with the low level of transpeptidase-mediated incorporation of unnatural D-amino acids of 0.2%-2. 8% (Hugonnet et al., 2016). Similar results were obtained for a PBP1B homolog from Pseudomonas aeruginosa (PBP1B Pa ) ( Figure S3B). We also tested E. coli PBP1A and could not find any evidence of GTase or TPase activity with this PBP with dansyl-GlcNAz-Lipid II ( Figure S3C). This indicates that the substrate specificity of the TPase activity of PBP1A differs from that of the other two tested PBPs. We next tested if we could incorporate GlcNAz into PG in situ by providing GlcNAz-CNP to E. coli cells expressing OleD. We hypothesized that GlcNAz-CNP is converted to UDP-GlcNAz by OleD, using the endogenous pool of UDP. UDP-GlcNAz can then be used to synthesize Lipid II, which in turn will be used for the synthesis of PG. To test this hypothesis, we expressed OleD in BL21(DE3) cells and added GlcNAz-CNP to the growth medium. In principle, the outer membrane of E.coli is permeable to a range of small molecules and sugars (Williams et al., 2011), thus it should be possible for BL21 cells to take up GlcNAz-CNP. Indeed, we observed that the color of the cell suspension turned more yellowish after incubation with cells expressing OleD ( Figure S4A), while this did not occur with control cells not expressing OleD. This indicates that 2-chloro-4-nitrophenol has been produced as a byproduct of the generation of UDP-GlcNAz inside the cells. Subsequently, the cells were subjected to copper (I)-catalyzed click reaction using Alexa Fluor 488-alkyne to test for the presence of accessible GlcNAz groups. Cells grown in the presence of GlcNAz-CNP were labeled, while cells grown in the absence of GlcNAz-CNP were not labeled (Figure 5A). Thus, this suggests that i) UDP-GlcNAz was produced inside the cells, ii) it is utilized by biosynthesis pathways, and iii) that at least some of the GlcNAz molecules were exported (to the periplasm) and subsequently labeled by the click reaction. The fluorescence appeared in patches on the cells, but also at other locations along the cell periphery. This pattern may point to PG-specific labeling. To test this possibility, we performed a Fö rster resonance energy transfer (FRET) experiment using fluorescently labeled vancomycin (Mohammadi et al., 2011). We reasoned that if the PG layer was fluorescently labeled with Alexa Fluor 488 attached to the incorporated GlcNAz, then binding of TMR-vancomycin to a nearby D-Ala-D-Ala should lead to FRET. To enable TMR-vancomycin to cross the outer membrane, we utilized EDTA to increase the permeability of the outer membrane of bacteria (MacNair and Brown, 2020). The occurrence of FRET is generally reflected by a decrease in fluorescence of the donor (Alexa Fluor 488) and an increase in fluorescence of the acceptor (TMR-vancomycin). Indeed, adding TMR-vancomycin to the Alexa Fluor 488-labeled cells lead to an FRET signal, indicating that at least part of the UDP-GlcNAz was used for the synthesis of Lipid II and nascent peptidoglycan which were then subsequently labeled ( Figure 5B).
Next, we aimed to obtain more direct evidence for the presence of GlcNAz in PG. For this, we isolated PG from cells expressing OleD and grown in the presence of GlcNAz-CNP. We digested the PG with the muramidase cellosyl to produce the disaccharide peptide subunits (muropeptides) and analyzed the muropeptides with nano-LC coupled to a mass spectrometer (Desmarais et al., 2014(Desmarais et al., , 2015. We were able to identify and confirm the presence of the GlcNAz-modified disaccharide tetrapeptide with a determined neutral mass of m/z = 981.436 (calculated m/z = 981.401, Figure 5C trace IV and V) that was not present in control Together, these results confirm that GlcNAz was specifically incorporated into bacterial PG. GlcNAz-CNP therefore can be converted into UDP-GlcNAz by heterologous OleD and the resulting precursor is used in the biosynthesis of PG. In our experiments, we did not observe azido MurNAc-containing compounds even though UDP-GlcNAc is a precursor of UDP-MurNAc in the bacterial cell. Most likely, the specificity of enzymes required for the UDP-MurNAc synthesis may be too high iScience Article and cannot use UDP-GlcNAz as a substrate. This labeling method could likely be applicable to other bacteria, provided that OleD can be heterologously expressed.
By expressing the heterologous glycosyltransferase OleD in bacteria, we could incorporate the unnatural amino sugar GlcNAz into the PG layer. Given the promiscuity of OleD for the sugar nucleotide donor (Gantt et al., 2011(Gantt et al., , 2013Williams et al., 2007), this labeling approach may find use in the modification of other sugars implicated in other biosynthetic processes. As we demonstrated here for PG, we envision that this method will be also applicable to introduce GlcNAc modified with a chemical handle or other sugar analogs in a range of hosts. Sugars such as glucose and galactose decorated with unique functionalities such as fluorophores, fluorine, or isotopes may be used this way for subsequent analysis by fluorescent microscopy, NMR, or MS.

Limitations of the study
Our study is limited to labeling bacteria (or other microorganisms) that express the OleD enzyme or homologs thereof. Lipid II labeled with a fluorophore at the N-acetylglucosamine cannot be used to study the glycosyl-transferase activity of PBPs.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  van Dam, V., Olrichs, N., and Breukink, E. (2009

Materials availability
Synthetic routes to all chemical compounds are described in Supplemental Information. Where available, these may be shared by the lead contact. Enzymes, reagents, and bacteria used for the biological studies were obtained from the commercial or internal sources described in the key resources table.
Data and code availability d All data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
E. coli Rosetta BL21(DE3) pLysS transformed with pET28a-OleD-TDP16 plasmid was used for the production of OleD and the in vivo incorporation of GlcNAz into the peptidoglycan layer. Experiments were conducted at 37 C unless otherwise noted. All information on alternate temperature conditions, as well as media conditions for experiments, can be found in the method details section.

Materials and instrumentation
All reagents employed were of American Chemical Society (ACS) grade or finer and were used without further purification unless otherwise stated. The compounds were purified by flash silica column chromatography (Merck, silica gel 60, 230$400 mesh). All reactions and fractions from column chromatography were monitored by thin-layer chromatography (TLC) using plates with or without a UV fluorescent indicator (Merck 60 F 254 , SO 2 ). Iodine (Merck) staining also was used for visualization of TLC. UDP-MurNAc-pentapeptide extract was obtained according to the previously reported method (Kohlrausch and Hö ltje, 1991). Undecaprenyl-phosphate was isolated from bay leaves and phosphorylated as described (Swiezewska et al., 1994;Danilov et al., 1989). Dansyl-lysine-lipid II was prepared as published (Breukink et al., 2003;Schwartz et al., 2002). iScience Article All NMR spectra were recorded at 400 MHz in Utrecht Institute for Pharmaceutical Sciences center with chemical shifts reported in parts per million (ppm) downfield relative to tetramethylsilane (TMS). 1 H NMR data are reported in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet), number of protons, and coupling constants (J) in Hertz (Hz). When appropriate, the multiplicity is preceded by br, indicating that the signal was broad. 13 C NMR spectra were recorded at 100 or 125 MHz with chemical shifts reported relative to CDCl 3 d 77.0. High-resolution mass spectrometry (HRMS) analysis was performed using an HRMS ESI-TOF or ESI-MS (Thermo Finnigan, LCQDECA XP) instrument. The fluorescent image of labeled bacteria was taken by Confocal Zeiss LSM 880 FAST AiryScan at the Biology Imaging Center at the Faculty of Science of Utrecht University.

Protein production and purification
We utilized a protocol based upon the previously published methods for the OleD enzyme (Gantt et al., 2011). A single colony of E. coli Rosetta BL21(DE3) pLysS transformed with pET28a-OleD-TDP16 plasmid, which was picked from the LB agar plate with 50 mg mL À1 kanamycin, was inoculated in 60 mL of LB with 50 mg mL À1 kanamycin at 37 C and in a shaker (200 rpm) overnight. The next morning, the starting culture was transferred into 6 L of LB medium supplemented with 50 mg mL À1 kanamycin and further grown in an incubator with shaking (200 rpm) at 37 C until the OD 600nm reached around 0.6. IPTG was added into the medium cultures to bring the final concentration of IPTG to 0.4 mM and the cultures were incubated at 25 C for around 16 h with shaking at 200 rpm. Bacteria were concentrated further by centrifugation at 6000g and 4 C for 15 min. The cell pellet was subsequently resuspended in 100 mL of lysis buffer (20 mM phosphate buffer, pH 7.4, 0.5 M NaCl, 10 mM imidazole, 50 mg mL À1 RNase, 10 mg mL À1 DNase and 0.25 mg mL À1 lysozyme) and lysed with a cell disruptor (Constant systems) for 5 cycles while cooling at 4 C. After the lysis, the disrupted bacteria cells were incubated on ice for a further 20 min to degrade peptidoglycan, DNA, and RNA. Cell debris was removed by centrifugation (38,000 g at 4 C for 15 min) and then the supernatant was applied to 6 mL of nickel resin equilibrated in wash buffer (20 mM phosphate buffer, pH 7.4, 0.3 M NaCl, 10 mM imidazole). The protein was incubated with the beads for 2 h on a digital tube roller at 4 C. Then the resin was applied to a column followed by a washing step with 150 mL of chilled wash buffer. Subsequently, the protein was eluted with 10 mL of elution buffer (20 mM phosphate buffer, pH 7.4, 0.3 M NaCl, 240 mM imidazole). Purified protein was concentrated and desalted using a PD-10 desalting column (GE Bioscience, USA), equilibrated with 50 mM Tris-HCl (pH 8.0), to provide 3 mL of the desired protein. The purity of the enzyme was determined by 10% SDS-PAGE, indicating the purity of more than 95%. The purified enzyme was stored at À80 C until use.

Chemoenzymatic synthesis of UDP-GlcNAz
Reactions were conducted at 37 C in 20 mL Tris-HCl buffer (50 mM, pH 8.0) with GlcNAz-CNP (5 mM), UDP (5 mM), MgSO 4 (25 mM), and OleD enzyme (125 mM). The reaction was monitored by TLC (2:1:1, n-butanol: acetic acid: H 2 O as eluent, A 254 nm ). Besides, the color of the reaction mixture turned yellowish from almost colorless as the reaction progressed. After 24 h, the reaction was stopped by adding 1 mL chloroform to precipitate the OleD enzyme. The reaction medium was centrifuged at 2,000g to remove the OleD enzyme. The aqueous upper phase was carefully collected and transferred into a new tube, followed by lyophilization. The dried yellowish residue was dissolved in 0.25 mL of Milli-Q water and the desired product was purified by silica gel column using n-butanol: acetic acid: water (2:1:1) as eluent. The product (UDP-GlcNAz, R f = 0.28, n-butanol: acetic acid: water = 2:1:1) was concentrated on a vacuum rotary evaporator and the residue was lyophilized to get a white powder (39 mg, yield 61%). iScience Article substrate (dansyl-GlcNAz-Lipid II or dansyl-lysine-Lipid II) in 50 mM Hepes at pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 0.05% TX-100. When indicated, reactions were supplemented with 10 mM unlabeled m-DAP-Lipid II, 50 mM moenomycin or 1 mM ampicillin. Reactions were incubated for 1 h (PBP1B Ec and PBP1B Pa ) or 2 h (PBP1A Ec ) at 37 C and then boiled for 5 min. Samples were vacuum-dried using a speed-vac desiccator and products were analyzed by Tris-Tricine SDS-PAGE as described previously (Herná ndez-Rocamora et al., 2021), with the exception that gels were visualized using a UV transilluminator.

Test for GlcNAz incorporation by intact bacteria
Overnight pre-cultured BL21 cells harboring pET28a-OleD-TDP16 were inoculated into fresh LB medium (with 50 mg mL À1 kanamycin) and were incubated until the OD 600nm was about 0.6. 1 mL of cells were collected by centrifugation at 6,000g for 2 min BL21 (OleD) were resuspended in 200 mL of LB medium (with 50 mg mL À1 kanamycin). GlcNAz-CNP (0.2%) and 0.4 mM IPTG were added to the cell samples. Cells were incubated at 37 C for 2 h and then collected ( FRET experiment of the Alexa fluor 488 labeled BL21 (pET28a-OleD-TDP16) and TMRvancomycin BL21 (pET28a-OleD-TDP16) cells were grown in LB with GlcNAz-CNP (0.2%) and labeled with Alexa fluor 488 via click chemistry. The labeled bacteria were resuspended into 1 mL of PBS (OD 600nm = 0.06) and EDTA was added into the suspension to bring the concentration to 1.7 mM. The bacteria were incubated with EDTA for 20 min at room temperature. Afterward, the bacterial suspension was scanned by a fluorometer (excited at 488 nm and data recorded between 500 nm and 700 nm). Then, TMR-Vancomycin (0.8 mM) was added to the suspension that was incubated for 20 min at room temperature. The data of the emission spectrum was recorded between 500 nm and 700 nm. The data of the control group (0.8 mM TMR-vancomycin in PBS with 1.7 mM EDTA) was recorded at the same conditions and fluorometer settings as the other sample. The FRET signal was plotted by the subtraction of the emission spectrum of TMR-Vancomycin from the emission spectrum of the mixture of Alexa fluor 488 labeled BL21 (pET28a-OleD-TDP16) and TMR-Vancomycin, which was recorded between 500 nm and 700 nm and excited at 488 nm.

PG preparation and analysis
BL21 pET28a-OleD-TDP16 cells were grown in 100 mL of LB medium in the presence or absence of 0.1% GlcNAz-CNP and the PG was isolated from these cells as previously published (Glauner, 1988). PG was digested overnight at 37 C with cellosyl in 20 mM ammonium formate (pH 4.8) and the samples were boiled for 10 min. The resulting muropeptides were reduced by adding 0.5 M ammonium hydroxide (pH 9.0, adjusted with formic acid) and $1 mg of solid tetramethylammonium borohydrate and incubating for 30 min at room temperature. The pH was adjusted to 3-4 with 20% formic acid prior to LC/MS analysis.
LC/MS analysis was performed as recently described (Taylor et al., 2021) with modifications as detailed below. For LC-MS/MS analysis 20 mL of muropeptide digest was acidified by addition of 1/10 th volume 10% formic acid (aq) and placed in an autosampler vial. 2 mL of acidified muropeptide was injected onto a microbore RP-HPLC column (ACE Ultracore 2.5 SuperC18, 0.5 3 150 mm) flowing at 15 mL min À1 delivered by a NanoAcquity UPLC system (Waters, UK). The column temperature was 35 C. Buffer A was composed of water containing 0.1% (v/v) formic acid. Buffer B was acetonitrile containing 0.1% (v/v) formic acid. The following elution gradient was used: starting at 0% buffer B, rising to 1.3% B at 3 min, then on to 2.5% B at 20 min, rising to 6.0% B at 35 min, the gradient was ramped to 10% B at 45 min, then to 50% B at 50 min, followed by 2 min at 85% B, and finally 12.5 min re-equilibration at 0% B. The total run time was 65 min.
The HPLC column eluate was directed to the mass spectrometer (Impact II QTof, Bruker) via an Apollo electrospray ion source (Bruker). The settings for the ion source were: capillary voltage and temperature settings of 3,200 V and 150 C respectively, together with a dry gas flow of 6 L min À1 and nebulizer pressure ll OPEN ACCESS iScience 25, 104753, August 19, 2022 iScience Article of 1.0 bar. MS data were acquired in positive ion mode over the range of 50$2,000 m/z at a spectral rate of 2 Hz. MS/MS acquisition was performed on the top five intense precursors in each scan (ions excluded after two MS/MS events). The preferred charge state range was set at +1 to +4 (undetermined charge states were excluded) and the sampling rate varied from 0.5 Hz at low ion counts (10,000) to 5.0 Hz for high-intensity ion counts (500,000). The resulting MS spectral data was opened for analysis using Compass DataAnalysis TM software (Bruker).
The synthesis of GlcNAz-CNP and NMR data Synthesis of compound 1 Glucosamine hydrochloric acid salt (3.0 g, 13.92 mmol, 1 eq) was dissolved in methanol (150 mL

Synthesis of compound 3
The compound 2 (54 mg, 0.1 mmol) was dissolved in dimethylformamide in a 50 mL round flask with a stirring bar. Sodium azide (65 mg, 1.0 mmol, 10 eq) was added to the reaction mixture before the reaction was kept stirring for 6 h at 60 C. The reaction mixture was poured into ice water (20 mL) and extracted with ethyl acetate 3 times (3 3 20 mL). The organic phase was collected and washed with water 3 times (3 3 20 mL). The organic phase was washed with saturated sodium bicarbonate solution (10 mL) before being washed with water 3 times (3 3 20 mL) again and dried with magnesium sulfate. The organic phase was filtered,

Synthesis of compound 4
To compound 3 (1.0 g, 18.4 mmol) in anhydrous methanol (30 mL) was added 25% (w/w) sodium methoxide (460 mL, 1 eq). After the reaction was stirred overnight, the solution turned a little yellowish the next morning. Amberlite IRC-50 resin was washed with methanol and added to the reaction mixture, which was stirred until the pH of the mixture reached 5 to 6. The mixture was filtered by an Amberlite IRC-50 resin desalting column and concentrated, purified by silica gel column chromatography with eluent (MeOH/DCM, from 1:10 to 1:5) to afford the off-white compound 4 (630 mg, 82.9%).

QUANTIFICATION AND STATISTICAL ANALYSIS
The fluorescence intensity in the FRET experiments was measured by Cary Eclipse Fluorescence Spectrometer. The raw data was collected by Cary Eclipse Scan Application 1.1. The experiments were repeated at least twice. The figure was produced with GraphPad Prism version 8.0.2 from the raw data.